Treatment of prostate cancer by inhibitors of NCAM2

ABSTRACT

The present invention is directed to a method of treating or detecting prostate cancer or breast cancer in a subject in need of such a treatment or detection by administering an inhibitor of NCAM2 in a pharmaceutically effective amount. The present invention also provides for a pharmaceutical composition comprising an inhibitor of NCAM2.

[0001] This application claims the benefit of priority of the U.S. provisional application U.S. Ser. No. 60/329,178 filed Oct. 11, 2001 and the U.S. provisional application U.S. Ser. No. 60/331,965, filed Nov. 21, 2001, each of which is incorporated by reference in its entirety.

FIELD OF THE INVENTION

[0002] This invention concerns methods for treating prostate cancer or breast cancer by inhibitors of RNCAM, preferably, NCAM2.

BACKGROUND OF THE INVENTION

[0003] Prostate cancer is the most common cancer in men with an estimated 244,000 cases in 1995 in the United States. It is the second leading cause among men who die from neoplasia with an estimated 44,000 deaths per year. Prompt detection and treatment is needed to limit mortality caused by prostate cancer. As described in W. J. Catalona, “Management of Cancer of the Prostate,” (New Engl. J. Med. 331(15): 996-1004 (1994)), the management of prostate cancer can be achieved by watchful waiting, curative treatment, and palliation.

[0004] A number of approaches have been developed to treat prostate cancer. Where prostate cancer is localized and the patient's life expectancy is 10 years or more, radical prostatectomy offers the best chance for eradication of the disease. Historically, the drawback of this procedure is that most cancers had spread beyond the bounds of the operation by the time they were detected. After surgery, if there are detectable serum prostate-specific antigen concentrations (PSA), persistent cancer is indicated. In many cases, prostate-specific antigen concentrations can be reduced by radiation treatment. However, this concentration often increases again within two years.

[0005] Cytotoxic chemotherapy is largely ineffective in treating prostate cancer. Its toxicity makes such therapy unsuitable for elderly patients. In addition, prostate cancer is relatively resistant to cytotoxic agents.

[0006] In view of the deficiency of the existing treatment approaches, it is of great significance to pursue new methods of treatment that particularly target the prostate tumor cells, such as anti-prostate tumor agents. The present invention has identified a novel tumor marker for prostate cancer, RNCAM, and is intended to use the inhibitors of this molecule as such anti-tumor agents.

[0007] NCAM (neural cell adhesion molecule) is the first molecule mediating cell-cell adhesion to be identified on the basis of functional criteria (Thiery, et al., J. Biol. Chem. 252: 6841-45 (1977)). Common features of these proteins are immunoglobulin-like domains in their NH2-terminal extracellular parts and sequences similar to fibronectin type III repeats carboxyl-terminal to them. Originally identified in chick embryo nervous system, NCAM is now known to be expressed in a great variety of tissues and cell types of all vertebrate species, and play important roles in axonal pathfinding and neurite outgrowth (Goridis, et al., Seminars in Cell Biology, Vol. 3, pp189-197 (1992); Baldwin et al., J. of Cellular Biochemistry 61: 502-513 (1996)).

[0008] Neural cell adhesion molecule 2 (NCAM2, NCBI protein accession number 4758764, see also Paoloni-Giacobino et al, Genomics 43, 43-51 (1997)) is a homologue of a murine protein called Rb-8 neural adhesion molecule (RNCAM, NCBI protein accession number 3334269, see also Alenius, M. and Bohm, S., J. Biol. Chem. 272, 26083-26083 (1997)). RNCAM is a novel molecule in NCAM family. Its amino acid sequence and protein expression pattern are different from the existing NCAM molecules. (Alenius, M. and Bohm, S., J. (1997)) The sequence of RNCAM predicted molecules having an extracellular region of 5 immunoglobulin C2-type domains followed by two fibronectin type III domains. Alternative splicing of the NCAM2 and RNCAM transcripts generate two isoforms: the long form containing a transmembrane domain and the short form containing a glycosylphosphatidylinositol-anchor attached to the membrane. The expression of RNCAM is restricted to the olfactory neurons in the brain and in the nasal vomeronasal organ. The transcript of RNCAM is not detectable in lung, gut, liver, heart, testis and kidney. The function of NCAM2 or RNCAM is not known, but the molecule may play a role in selective axon projection.

[0009] So far no investigation has been conducted to explore the possible therapeutic application of inhibitors of RNCAM (NCAM2) in prostate cancer treatment. This invention is directed to methods for treating or preventing prostate cancer or breast cancer by using the inhibitors of RNCAM, preferably, NCAM2.

SUMMARY OF THE INVENTION

[0010] The present invention is directed to a method of treating or preventing prostate cancer or breast cancer in a subject in need of such a treatment by administering an inhibitor of NCAM2 in a pharmaceutically effective amount.

[0011] The present invention also provides a pharmaceutical composition comprising an inhibitor of NCAM2.

[0012] The present invention further provides for a method of detecting a cancer derived from a type of cells comprising detecting presence of NCAM2 in said type of cells.

[0013] Preferably, the present invention provides for a method of detecting prostate cancer or breast cancer comprising detecting presence of NCAM2 in prostate cells or breast cancer cells of a subject in need of the detecting.

[0014] The present invention also provides for a method of inhibiting growth of a cancer cell comprising contacting an inhibitor of NCAM2 with said cancer cell. Preferably, said cancer cell is a prostate cancer cell or a breast cancer cell.

[0015] The cancers can be treated or detected by the methods of the present invention include, but are not limited to, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and leukemia.

BRIEF DESCRIPTION OF THE DRAWINGS

[0016]FIG. 1. Flow chart summarizing how the tumor-specific hybridomas were obtained from the LNCaP lipid raft immunization.

[0017]FIG. 2. Antigen grouping by immunoprecipitation. ¹²⁵I labeled LNCaP lysate was incubated individually with 20 hybridoma supernatants (see Table 1). Antibody-antigen complexes were captured by Gamma Bind Plus Sepharose and analyzed by SDS-PAGE. Lane 1, P1-42; Lane 2, P2-23; Lane 3, P3-53; Lane 4, P4-79; Lane 5, P6-49; Lane 6, P9-65; Lane 7, P8-2; Lane 8, P8-11; Lane 9, P8-14; Lane 10, P8-35; Lane 11, P8-74; Lane 12, P9-32; Lane 13, P9-64; Lane 14, P10-2; Lane 15, P10-28, Lane 16, P10-29; Lane 17, P10-62; Lane 18, P10-70; Lane 19, P10-82; and Lane 20, P12-22. Molecular weight standards (MW) are in kD.

[0018]FIG. 3. Anti-NCAM2 inhibits LNCaP cell proliferation. LNCaP cells (20,000 cells/well) were plated into a 96 well tissue culture plate. After cells were allowed to grow undisturbed for two days, 4 different NCAM2-specific antibodies (5 μg/ml) were added and incubated with the cells for 24 hours. AlamarBlue reagent was added to assess cell proliferation. Fluorescence was detected at λex=530 nm, λem=590 nm. Data are expressed as the mean +/− SEM of 4 replicates.

[0019]FIG. 4. Anti-NCAM2 inhibits LNCaP colony formation in soft agar. LNCaP cells were plated in soft agar and treated with 4 different NCAM2-specific antibodies (5 μg/ml) for up to 20 days. Colonies were counted under an inverted phase-contrast microscope and a group of 10 or more cells were counted as a colony.

[0020]FIG. 5. Table 1 shows the reactivity profiles of 20 anti-LNCaP lipid raft hybridomas that showed limited binding to other cancer cell lines.

[0021]FIG. 6. Table 3 shows the reactivity profiles of 1 anti-LNCaP lipid raft hybridomas that showed broad binding to other cancer cell lines.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0022] Definitions:

[0023] As used herein, the term “antibody” or “immunoglobulin” refers to a protein consisting of one or more polypeptides substantially encoded by immunoglobulin genes. The recognized immunoglobulin genes include the kappa, lambda, alpha, gamma (IgG₁, IgG₂, IgG₃, IgG₄), delta, epsilon and mu constant region genes, as well as the myriad immunoglobulin variable region genes. Full-length immunoglobulin “light chains” (about 25 Kd or 214 amino acids) are encoded by a variable region gene at the NH2-terminus (about 110 amino acids) and a kappa or lambda constant region gene at the COOH—terminus. Full-length immunoglobulin “heavy chains” (about 50 Kd or 446 amino acids), are similarly encoded by a variable region gene (about 116 amino acids) and one of the other aforementioned constant region genes, e.g., gamma (encoding about 330 amino acids).

[0024] One form of immunoglobulin constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of immunoglobulin chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions. In addition to antibodies, immunoglobulins may exist in a variety of other forms including, for example, Fv, Fab, and (Fab′)₂, as well as bifunctional hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference). (See, generally, Hood et al., “Immunology”, Benjamin, N.Y., 2nd ed. (1984), and Hunkapiller and Hood, Nature, 323, 15-16 (1986), which are incorporated herein by reference).

[0025] The preferred antibody is the monoclonal antibody that binds to or neutralizes RNCAM, preferably NCAM2.

[0026] By “a pharmaceutically effective” amount of a drug or pharmacologically active agent or pharmaceutical formulation is meant a nontoxic but sufficient amount of the drug, agent or formulation to provide the desired effect.

[0027] A “subject,” “individual” or “patient” is used interchangeably herein, which refers to a vertebrate, preferably a mammal, more preferably a human.

[0028] The term “inhibit growth of cancer (tumor) cells” refers to any action that may decrease the growth of a cancer cell. The inhibition may reduce the growth rate or the size of cancer cells, or inhibit or prevent proliferation, growth, or migration of cancer cells. The inhibition may inhibit the colony formation of cancer cells due to the inhibition of anchorage-independent growth. Preferably, such an inhibition at the cellular level may reduce the size, deter the growth, reduce the aggressiveness, or prevent or inhibit metastasis of a tumor in a patient.

[0029] The term “colony formation” refers to the number of cancer (tumor) cell colonies formed due to the anchorage-independent cancer (tumor) cell growth. A variety of methods can be used to measure the “colony formation”, such as counting the number of the formed colonies (see Examples).

[0030] The term “lipid raft” refers to a lipid raft or a portion thereof in a clustered state or a non-clustered state, including “lipid raft”, “clustered lipid rafts”, and “DRM”, each of which has been described in detail in Simons, K., et al., Nature Reviews/Molecular Cell Biology: Vol. 1 pp 31-39 (2000). In particular, “lipid raft” contains a given set of proteins that can change size and composition in response to intra- or extracellular stimuli. This favors specific protein-protein interactions, resulting in the activation of signally cascade. Sometimes, the lipid rafts may be clustered together. It has been reported that clustering is used both artificially and physiologically to trigger signally cascades. DRMs (detergent-resistant membranes) are the rafts that remain insoluble after treatment on ice with detergents, such as Triton X-100 or NP-40. They are believed to be non-native aggregated rafts.

[0031] The term “epitope” includes any protein determinant capable of specific binding to an immunoglobulin or an antibody. Epitopic determinants usually consist of active surface groupings of molecules such as amino acids or sugar side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Two antibodies are said to bind to the same epitope of a protein if amino acid mutations in the protein that reduce or eliminate binding of one antibody also reduce or eliminate binding of the other antibody, and/or if the antibodies compete for binding to the protein, i.e., binding of one antibody to the protein reduces or eliminates binding of the other antibody.

[0032] The term “derived from” means “obtained from” or “produced by”.

[0033] The term “genetically altered antibodies” means antibodies wherein the amino acid sequence has been varied from that of a native antibody. Because of the relevance of recombinant DNA techniques to this invention, one need not be confined to the sequences of amino acids found in natural antibodies; antibodies can be redesigned to obtain desired characteristics. The possible variations are many and range from the changing of just one or a few amino acids to the complete redesign of, for example, the variable or constant region. Changes in the constant region will, in general, be made in order to improve or alter characteristics, such as complement fixation, interaction with membranes and other effector functions. Changes in the variable region will be made in order to improve the antigen binding characteristics.

[0034] The term “humanized antibody” or “humanized immunoglobulin” refers to an immunoglobulin comprising a human framework, at least one and preferably all complimentarity determining regions (CDRs) from a non-human antibody, and in which any constant region present is substantially identical to a human immunoglobulin constant region, i.e., at least about 85-90%, preferably at least 95% identical. Hence, all parts of a humanized immunoglobulin, except possibly the CDRs, are substantially identical to corresponding parts of one or more native human immunoglobulin sequences. See, e.g. Queen et al., U.S. Pat. Nos. 5,5301,101; 5,585,089; 5,693,762; and 6,180,370 (each of which is incorporated by reference in its entirety).

[0035] The term “chimeric antibody” refers to an antibody in which the constant region comes from an antibody of one species (typically human) and the variable region comes from an antibody of another species (typically rodent).

[0036] The present invention provides a method of treating prostate cancer or breast cancer.

[0037] In a preferred embodiment of the present invention, an inhibitor of RNCAM is administered to a subject in need of such a treatment in a pharmaceutically effective amount. Preferably, said RNCAM is a human NCAM2.

[0038] The present invention also provide for a method of inhibiting growth of a cancer cell comprising contacting an inhibitor of RNCAM, preferably NCAN2 with said cancer cell. Preferably, said cancer cell is a prostate cancer cell or a breast cancer cell. Preferably, said inhibiting reduces the proliferation of a cancer cell by more than 10%, more preferably, by more than 30%, even more preferably, by about 39%. In addition, said inhibiting can reduce the colony formation of a cancer cell, preferably, a prostate cancer cell, by more than 20%, more preferably, by more than 40%, even more preferably, by about 42%.

[0039] Preferably, the inhibitor is a protein.

[0040] Preferably, the protein directly interacts with RNCAM, preferably NCAM2.

[0041] Preferably, the protein binds to RNCAM, preferably NCAM2.

[0042] More preferably, the inhibitor is an anti-RNCAM (preferably anti-NCAM2) antibody or antibody fragment thereof. More preferably, said antibody or the fragment thereof neutralizes RNCAM (preferably, NCAM2) biological activity.

[0043] In addition, the inhibitor can also be a compound that inhibits the biological activities of RNCAM (preferably, NCAM2). More examples include molecules that may interact with RNCAM (preferably, NCAM2) signaling pathway and down-regulates the activity of RNCAM (preferably, NCAM2). The understanding of NCAM signaling pathway provides the useful information for the search of this kind of compounds. It has been reported that antagonists of calcium channels can inhibit NCAM activities (NCAM-dependent neurite outgrowth). In additions, growth factors such as FGF may regulate the activities of NCAM (Baldwin et al.). Such understanding will provide the starting point for the search of the molecules down-regulating NCAM2. The inhibitors can also be mutant RNCAM (preferably, NCAM2) derived from a wild-type RNCAM (preferably, NCAM2) by terminal truncation or amino acid substitution. Preferably such mutant NCAM2 can retain the binding of the NCAM2 to it signaling molecule but lose the ability of triggering the signaling cascade of NCAM2 and thus blocking the biological activity of NCAM2. The suitable compounds can be sought by using the conventional techniques known to a skilled artisan in the field of molecular biology, organic chemistry and biochemistry.

[0044] In addition, the inhibitor can inhibit the protein expression of RNCAM (preferably, NCAM2). RNCAM expression can be regulated at the level of alternative splicing of RNCAM mRNA, or by a transcription factor (Gorisdis, C., et al.). The inhibitor in the present invention includes the down-regulating molecules at each level. The inhibitor can also be a nucleic acid, including but not limited to, an anti-sense nucleic acid of the nucleic acid sequence encoding part or full or having substantial sequence similarity of RNCAM (preferably, NCAM2). The DNA sequence of NCAM2 is known in the art. Subsequently, anti-sense nucleic acid probe of DNA of NCAM2, and the optimal condition of the anti-sense blocking can be developed by using the related techniques known to a skilled artisan in the field of molecular biology.

[0045] The present invention also provides for a pharmaceutical composition comprising an inhibitor of RNCAM (preferably, NCAM2). The pharmaceutical composition can further comprise a pharmaceutically acceptable carrier. Preferably, such a composition comprises anti-RNCAM antibodies, more preferably, anti-NCAM2 antibodies.

[0046] The effective treatment of prostate cancer by the inhibitors of RNCAM (preferably, NCAM2) includes various stages, such as androgen-dependent prostate cancer and androgen-independent prostate cancer.

[0047] The anti-NCAM2 antibodies may be in a polyclonal or monoclonal form and may bind to any epitope or subunit of NCAM2). Preferably, the anti-NCAM2 antibody is an isolated monoclonal antibody. Anti-NCAM2 antibodies of all species of origins are included. Non-limiting exemplary anti-NCAM2 antibodies include antibodies derived from human, chicken, goats, and rodents (e.g., rats, mice, hamsters and rabbits), including transgenic rodents genetically engineered to produce human antibodies (see, e.g., Lonberg et al., WO93/12227 (1993) and Kucherlapati, et al., WO91/10741 (1991)), which are herein incorporated by reference in their entirety).

[0048] Preferably, the anti-NCAM2 antibody is an isolated monoclonal antibody that binds to or neutralizes NCAM2.

[0049] Preferably, the antibodies can inhibit the proliferation of a cancer cell by at least 10%, preferably at least 15%, and more preferably at least 30% or 35%, or as high as 39%. Preferably, such a cancer is a malignant solid tumor, more preferably, a prostate cancer.

[0050] Preferably, the antibodies inhibit prostate cancer cell colony formation, more preferably, by more than 20% or by more than 40%.

[0051] In one aspect of the present invention, the anti-NCAM2 antibodies can also bind to an isolated lipid raft. Preferably, the isolated lipid raft is derived from a cancer cell, and more preferably a prostate cancer cell. The present invention for the first time discovers the association of NCAM2 with lipid rafts of cancer cells. Immunization of a host animal, for example mouse, with the lipid rafts obtained from prostate cancer cells gives rise to hybridoma antibodies that bind to NCAM2 as well as the lipid raft. Accordingly, NCAM2 exists as a component of the lipid rafts in cancer cells. These hybridoma antibodies include, but are not limited to, the antibodies produced by the hybridoma cell line P3-53, P9-64, P10-28, or P10-29.

[0052] Preferably, the present invention provides a hybridoma cell line P9-64 deposited with American Type Culture Collection (ATCC) as accession number PTA-4734. The deposit of this hybridoma cell line (PTA-4734) was received by American Type Culture Collection (ATCC) on Oct. 1, 2002.

[0053] More preferably, the present invention provides a monoclonal antibody produced by the hybridoma cell line having an ATCC Accession Number PTA-4734.

[0054] The polyclonal forms of these antibodies can be produced by immunization of host animals by NCAM2. The polyclonal antibodies are secreted into the bloodstream and can be recovered using known techniques. Purified forms of these antibodies can, of course, be readily prepared by standard purification techniques, preferably including affinity chromatography with Protein A, anti-immunoglobulin, or the antigen itself. In any case, in order to monitor the success of immunization, the antibody levels with respect to the antigen in serum will be monitored using standard techniques such as ELISA, RIA and the like.

[0055] The monoclonal antibodies can be produced by conventional hybridoma methodology known in the art. In particular, after the immunization with NCAM2, the host animal may be sacrificed and the lymphocytes of said animal are isolated. The lymphocytes can produce or be capable of producing antibodies that specifically bind to the protein used for immunization. Lymphocytes then are fused with myeloma cells using suitable fusing agents to form hybridomas cells that produce the desired monoclonal antibody.

[0056] The antibodies can also be produced by using the method of lipid raft immunization, which is disclosed in Examples and U.S. Ser. No. 60/331,965, hereby incorporated by reference in its entirety. The present invention discovered the association of NCAM2 with the lipid rafts derived from cancer cells. Accordingly, the anti-NCAM2 antibodies can be produced by immunizing host animals with isolated lipid rafts from cancer cells.

[0057] Antibodies useful in the present invention also may be made using phage display methods (see, e.g., Dower et al., WO91/17271 and McCafferty et al., WO92/01047, which are herein incorporated by reference in their entirety.

[0058] The present invention also includes genetically altered anti-NCAM2 antibodies that are functionally equivalent to above antibodies and antibody fragments. Modified antibodies providing improved stability and/or therapeutic efficacy are preferred. Examples of modified antibodies include those with conservative substitutions of amino acid residues, and one or more deletions or additions of amino acids which do not significantly deleteriously alter the antigen binding utility. Substitutions can range from changing or modifying one or more amino acid residues to complete redesign of a region as long as the therapeutic utility is maintained. Antibodies of this invention can be can be modified post-translationally (e.g., acetylation, and phosphorylation) or can be modified synthetically (e.g., the attachment of a labeling group). Fragments of these modified antibodies that retain the binding specificity can also be used.

[0059] The genetically altered antibodies also include chimeric antibodies that bind to and neutralize NCAM2. Preferably, the chimeric antibodies comprise a variable region derived from a mouse or rat and a constant region derived from a human so that the chimeric antibody has a longer half-life and is less immunogenic when administered to a human subject. The method of making chimeric antibodies is known in the art.

[0060] Preferably, the genetically altered antibodies used in the present invention include humanized antibodies that bind to or neutralize NCAM2. More preferably, said humanized antibody comprising CDRs of a mouse donor immunoglobulin and heavy chain and light chain frameworks of a human acceptor immunoglobulin. The method of making humanized antibody is disclosed in U.S. Pat. Nos.: 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 each of which is incorporated by reference in its entirety.

[0061] Anti-NCAM2 fully human antibody is also included in the present invention. Fully human antibody against NCAM2 is produced by a variety of techniques. First, trioma methodology may be used to develop the fully human antibody. The basic approach and an exemplary cell fusion partner, SPAZ-4, for use in this approach have been described by Oestberg et al., Hybridoma 2:361-367 (1983); Oestberg, U.S. Pat. No. 4,634,664;and Engleman et al., U.S. Pat. No. 4,634,666 (each of which is incorporated by reference in its entirety for all purposes).

[0062] Human antibodies against NCAM2 can also be produced from non-human transgenic animals having transgenes encoding at least a segment of the human immunoglobulin locus. Usually, the endogenous immunoglobulin locus of such transgenic animals is functionally inactivated. Preferably, the segment of the human immunoglobulin locus includes unrearranged sequences of heavy and light chain components. Both inactivation of endogenous immunoglobulin genes and introduction of exogenous immunoglobulin genes can be achieved by targeted homologous recombination, or by introduction of YAC chromosomes. The transgenic animals resulting from this process are capable of functionally rearranging the immunoglobulin component sequences, and expressing a repertoire of antibodies of various isotypes encoded by human immunoglobulin genes, without expressing endogenous immunoglobulin genes. The production and properties of animals having these properties are described in detail by, e.g., Lonberg et al., WO 93/12227 (1993); Kucherlapati, WO 91/10741 (1991) (each of which is incorporated by reference in its entirety).

[0063] Phage display methods can also be used for obtaining human anti-NCAM2 antibodies. The methods include the steps of screening a DNA library from human B cells according to the general protocol outlined by Huse et al., Science 246:1275-1281 (1989). Antibodies binding to NCAM2 or a fragment thereof are selected. Sequences encoding such antibodies (or binding fragments) are then cloned and amplified. The protocol described by Huse is rendered more efficient in combination with phage-display technology. See, e.g., Dower et al., WO 91/17271 and McCafferty et al., WO 92/01047 (each of which is incorporated by reference in its entirety).

[0064] The fragments of the above-described anti-NCAM2 antibodies, which retain the binding specificity to NCAM2, are also included in the present invention. Examples include, but are not limited to, the heavy chains, the light chains, and the variable regions as well as the truncated chains (truncated at the carboxyl end), which is particularly useful for immunoscintigraphic procedures. Examples of truncated chains include, but are not limited to Fab fragment (consisting of the VL, VH, CL and CH1 domains): the Fd fragment (consisting of the VH and CH1 domains); the Fv (consisting of VL and VH domains of a single arm of an antibody); dab fragment (consisting of a VH domain); isolated CDR regions; F(ab′)₂ fragment, a bivalent fragment (comprising two Fab fragments linked by a disulphide bridge at the hinge region). The truncated chains can be produced by conventional biochemistry techniques, such as enzyme cleavage, or recombinant DNA techniques, each of which is known in the art.

[0065] The genes of the antibody fragments may be fused to functional regions from other genes (e.g., enzymes, U.S. Pat. No. 5,004,692, which is incorporated by reference in its entirty) to produce fusion proteins (e.g., immunotoxins) or conjugates having novel properties.

[0066] When used therapeutically, the antibodies disclosed herein may be used in unmodified form or may be modified with an effector moiety that delivers a toxic effect, such as a drug, cytotoxin (preferably, a protein cytotoxin or a Fc domain of the monoclonal antibodies), radionuclide, etc (see, e.g., U.S. Pat. No. 6,086,900, which is hereby incorporated by reference in its entirety).

[0067] Preferably, a pharmaceutical composition of the present invention comprises the use of the subject antibodies in immunotoxins. Conjugates that are immunotoxins including conventional antibodies have been widely described in the art. The toxins may be coupled to the antibodies by conventional coupling techniques or immunotoxins containing protein toxin portions can be produced as fusion proteins. The conjugates of the present invention can be used in a corresponding way to obtain such immunotoxins. Illustrative of such immunotoxins are those described by Byers, B. S. et al. Seminars Cell Biol 2:59-70 (1991) and by Fanger, M. W. et al. Immunol Today 12:51-54 (1991).

[0068] A variety of cytotoxic agents are suitable for use in immunotoxins. Cytotoxic agents can include radionuclides, such as Iodine-131 or other isotopes of iodine, Yttrium-90, Rhenium-188, and Bismuth-212 or other alpha emitters; a number of chemotherapeutic drugs, such as vindesine, methotrexate, adriamycin, and cisplatinum; and cytotoxic proteins such as ribosomal inhibiting proteins like pokeweed antiviral protein, Pseudomonas exotoxin A, ricin, diphtheria toxin, ricin A chain, etc., or an agent active at the cell surface, such as the phospholipase enzymes (e.g., phospholipase C). (See, generally, “Chimeric Toxins,” Olsnes and Phil, Pharmac. Ther., 25, 355-381 (1982), and “Monoclonal Antibodies for Cancer Detection and Therapy,” eds. Baldwin and Byers, pp. 159-179, 224-266, Academic Press (1985), all of which are incorporated herein by reference.)

[0069] The delivery component of the immunotoxin will include the antibodies described herein, including chimeric antibodies, humanized antibodies, and human antibodies of the present invention. Intact immunoglobulins or their binding fragments, such as Fab, are preferably used. Typically, the antibodies in the immunotoxins will be of the human IgM or IgG isotype, but other mammalian constant regions may be utilized as desired.

[0070] There are various methods of administering the inhibitors. The inhibitor may be administered to a patient intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, topical, inhalation routes, or other delivery means known to the people skilled in the art.

[0071] Preferably, pharmaceutical compositions of the present invention are useful for parenteral administration, i.e., subcutaneously, intramuscularly and particularly, intravenously. The compositions for parenteral administration commonly comprise a solution of the inhibitor, preferably the antibody, or a cocktail thereof dissolved in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers can be used, e.g., water, buffered water, 0.4% saline, 0.3% glycine and the like. These solutions are sterile and generally free of particulate matter. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, toxicity adjusting agents and the like, for example sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, histidine and arginine. The concentration of the inhibitors (preferably antibodies) in these formulations can vary widely, i.e., from less than about 0.01%, usually at least about 0.1% to as much as 5% by weight and are selected primarily based on fluid volumes, and solubilities in accordance with the particular mode of administration selected.

[0072] Thus, a typical pharmaceutical composition for injection could be made up to contain 1 ml sterile buffered water, and 1-100 mg of an inhibitor. A typical composition for intravenous infusion can be made up to contain 250 ml of sterile Ringer's solution, and 10 mg of the inhibitor, such as anti-NCAM2 antibody. Actual methods for preparing parentally administrable compositions are known or apparent to those skilled in the art and are described in more detail in, for example, Remington's Pharmaceutical Science (15th Ed., Mack Publishing Company, Easton, Pa., 1980), which is incorporated herein by reference.

[0073] The present invention provides for a pharmaceutical composition comprising an inhibitor of NCAM2, preferably, an antibody that binds to or neutralizes NCAM2.

[0074] For the purpose of treatment of disease, the appropriate dosage of the above inhibitors will depend on the severity and course of disease, the patient's clinical history and response, the toxicity of the inhibitors, and the discretion of the attending physician. The inhibitors are suitably administered to the patient at one time or over a series of treatments. The initial candidate dosage may be administered to a patient. The proper dosage and treatment regime can be established by monitoring the progress of therapy using conventional techniques known to the people skilled of the art.

[0075] The amount of active ingredients that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors, including the activity of the specific inhibitor employed, the age, body weight, general health, sex, diet, time of administration, route of administration, and rate of excretion, drug combination and the severity of the particular disease undergoing therapy, and can be determined by those skilled in the art.

[0076] The compositions can be administered for prophylactic and/or therapeutic treatments, comprising preventing, inhibiting, reversing tumor cell proliferation, and/or reducing tumor size. An amount adequate to accomplish the desired effect without toxic effect is defined as a “pharmaceutically effective amount” and will generally range from about 0.01 to about 100 mg of antibody per dose.

[0077] Additionally, the inhibitors can be utilized alone in substantially pure form, or together with chemotherapeutic agents, as are known to those of skill in the art (see, e.g., Cancer: Principles and Practice of Oncology, 5^(th) ed., Devita et al., Lippincott-Ravel Publishers, 1997). Other therapies that may be used in conjunction with treatment with the antibodies include administration of anti-sense nucleic acid molecules or biologicals, such as additional therapeutic antibodies, as well as radiation and/or surgery (see, e.g., WO0034337). Thus, the treatment of the present invention is formulated in a manner allowing it to be administered serially or in combination with another agent for the treatment of cancer.

[0078] Antibodies disclosed herein are useful in diagnostic and prognostic evaluation of diseases and disorders, particularly cancers associated with NCAM2 expression. At each stage of disease, monoclonal antibodies may be used to improve diagnostic accuracy and facilitate treatment decisions. Unlike standard diagnostic methods for tumors and cancer, such as computed topographic (CT) scans, which depend on a change in size or architecture of organs or lymph nodes, labeled monoclonals can detect abnormal cells at an early stage, because of their expression of tumor antigens, such as NCAM2. Once cancer is diagnosed, accurate staging is important in deciding on the most appropriate therapy. Later, during follow-up of surgery, rising serum levels of tumor antigens may indicate recurrence before it can be detected by conventional methods.

[0079] Methods of diagnosis can be performed in vitro using a cellular sample (e.g., blood sample, lymph node biopsy or tissue) from a patient or can be performed by in vivo imaging.

[0080] In particular embodiments, the present invention provides a antibody conjugate wherein the antibodies of the present invention is conjugated to a diagnostic imaging agent. Compositions comprising the antibodies of the present invention can be used to detect NCAM2, for example, by radioimmunoassay, ELISA, FACS, etc. One or more labeling moieties can be attached to the humanized immunoglobulin. Exemplary labeling moieties include radiopaque dyes, radiocontrast agents, fluorescent molecules, spin-labeled molecules, enzymes, or other labeling moieties of diagnostic value, particularly in radiologic or magnetic resonance imaging techniques.

[0081] The present invention also provides for a method of detecting prostate cancer or breast cancer, comprising detecting the presence of NCAM2 in the prostate cells or breast cells of a subject in need of such detection. Since our data show that NCAM2 is differentially expressed in the prostate tumor cells or breast cancer cells but not normal prostate cells, an antibody against NCAM2 can be used as a bio-marker for detecting the prostate cancer or breast cancer.

[0082] The present invention also provides for a diagnostic kit comprising anti-NCAM2 antibodies. Such a diagnostic kit further comprises a packaged combination of reagents in predetermined amounts with instructions for performing the diagnostic assay. Where the antibody is labeled with an enzyme, the kit will include substrates and co-factors required by the enzyme. In addition, other additives may be included such as stablizers, buffers and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents that substantially optimize the sensitivity of the assay. Particularly, the reagents may be provided as dry powders, usually lyophilized, including excipients that, on dissolution, will provide a reagent solution having the appropriate concentration.

[0083] The inhibitors of the present invention may also be employed for the inhibition of cancer cell growth, or for the treatment of other types of cancer or neoplasm or malignant tumors found in mammals, including carcinomas and sarcomas. Examples of cancers are cancer of the brain, breast, cervix, bladder, colon, head & neck, kidney, lung, non-small cell lung, melanoma, mesothelioma, ovary, sarcoma, stomach, uterus, and Medulloblastoma. Preferably, the inhibitors may be employed to detect or treat disorders including, but not limited to, Hodgkin's Disease, Non-Hodgkin's Lymphoma, multiple myeloma, neuroblastoma, breast cancer, ovarian cancer, lung cancer, rhabdomyosarcoma, primary thrombocytosis, primary macroglobulinemia, small-cell lung tumors, primary brain tumors, stomach cancer, colon cancer, malignant pancreatic insulanoma, malignant carcinoid, urinary bladder cancer, premalignant skin lesions, testicular cancer, lymphomas, thyroid cancer, neuroblastoma, esophageal cancer, genitourinary tract cancer, malignant hypercalcemia, cervical cancer, endometrial cancer, adrenal cortical cancer, and leukemia.

[0084] Though the inhibitors of the present invention are primarily concerned with the treatment of human subjects, they may also be employed for the treatment of other mammalian subjects such as dogs and cats for veterinary purposes.

[0085] The following examples are offered by way of illustration and not by way of limitation. The disclosure of all citations in the specification is expressly incorporated herein by reference.

EXAMPLES Example 1

[0086] This example describes identifying the anti-prostate tumor agent by the immunization of lipid rafts from LNCaP cell lines.

[0087] Materials and Methods

[0088] a. Lipid Raft Preparation

[0089] Lipid rafts were prepared as described in Green et al, J. Cell Biol. 146, 673-682 (1999). Briefly, cells (8.0×10⁶ cells/sample) were lysed in 0.1% vol/vol Brij-58, 20 mM Tris HCl, pH 8.2, 140 mM NaCl, 2 mM EDTA, 25 μg/ml aprotinin, 25 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride for 10 minutes on ice. Cells were homogenized using 10 strokes of a Dounce homogenizer, then lysed 20 minutes more on ice. The resulting lysate was adjusted to 40% wt/wt sucrose and applied onto a 60% wt/wt sucrose cushion. A sucrose step-gradient consisting of 25% wt/wt sucrose and 5% wt/wt sucrose were layered on top of the lysate. Gradients were centrifuged 18 hours at 170,000×g at 4° C. in a SW55 rotor. Fractions (0.2 ml) were taken from the top of the gradient. Lipid rafts float to the interface of the 25% and 5% sucrose layers (Fractions 7 and 8). The amount of protein in each fraction was determined using the BCA Protein Assay Kit. Protein was concentrated by centrifugation at 2000×g in Vivaspin 6 PES membrane columns (molecular weight cut off=10,000 kDa).

[0090] b. Lipid Raft Immunization

[0091] Lipid raft proteins (approximately 5 μg) were mixed together with 50 μL Ribi®, and then injected into the foot pad of a BALB/c mouse. Mice were boosted with 50 μL of lipid raft proteins in Ribi® on day 7 and day 14. Three days after the last boost, the mice were sacrificed and the hind leg lymph node was harvested. The lymph node was washed in pre-warmed DMEM and then ground using a Dounce homogenizer. After 5 gentle strokes, the cell suspension was removed into the plastic tube. This process was repeated four more times, each time adding 5 ml of fresh DMEM. The lymphocytes were pooled and washed 3 times in DMEM. The lymphocytes were mixed with the appropriate number of pre-washed fusion partner NSO/BCL-2 (NS0 transfectant expressing the mouse BCL-2 cDNA) to yield ratio of 2-3 lymphocytes for every 1 NS0. The mixture was pelleted and warmed at 37° C. for 1 min. Pre-warmed 50% PEG was slowly added onto the pellet and then cells were centrifuged at 300×g for 3 minutes at room temperature. Five mL DMEM was added and then 10 mL DMEM with 10% FBS and 1% P/S was added. The cells were then centrifuged 5 minutes at 300×g at room temperature. The pellet was resuspended in HAT selection medium (DMEM with 20% fetal bovine serum, 2 mM glutamine, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 0.1 mM sodium hypoxanthine, 16 μM thymidine, 20 μM aminopterin, 2×Origen cloning factor, 10 mM HEPES, 50 μM beta-mercaptoethanol, 0.2 units/mL penicillin, 0.2 μg/mL streptomycin) to yield 0.25×10⁶ lymphocytes/mL. Cells were aliquoted into ten 96-well flat bottom plates at 200 μL per well for the selection of hybridomas.

[0092] c. Flow Cytometry Screening

[0093] Flow cytometry was used to screen hybridoma supernatants for the presence of cell surface binding antibodies. T he cells (2×10⁵) were resuspended in 100 μL ice cold PBS with 10 μL tissue culture supernatant on ice for 1 hour. After extensive washing, cells were incubated with phycoerythrin-conjugated goat antibodies specific for mouse IgG for 30 minutes on ice. Cells were washed again and cell surface bound antibody was detected using a Becton Dickenson FACScan. Additionally, hybridoma supernatants were similarly screened on many cancer cell lines or whole blood to test for specificity.

[0094] d. Affinity Purification of Antigen

[0095] Cells (5×10⁸) were lysed in 1% vol/vol NP-40, 0.5% wt/vol deoxycholate, 20 mM Tris HCl, pH 8.2, 150 mM NaCl, 1 mM EDTA, 25 μg/ml aprotinin, 25 μg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride for 1 hour on ice with frequent mixing. Lysate was centrifuged for 20 minutes at 300×g to remove nuclei and debris. Antigens were purified by standard hybridoma affinity chromatography techniques as described in Hill et al, J. Immunol. 152, 2890-2898 (1994).

[0096] e. Antigen Grouping

[0097] Cells (2×10⁷) were cell surface iodinated as described (Landolfi and Cook, Mol. Immunol. 23, 297-309 (1986)). Cells were then lysed in 1% NP-40, 0.5% deoxycholate, 50 mM Tris-HCl , pH 7.4, 150 mM NaCl, 2 mM EDTA, 10 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 mM PMSF for 1 hour on ice. Cell lysate was centrifuged at 14,000×g for 5 minutes to remove nuclei and debris. Cell lysate was pre-cleared with rotation by incubation with Gamma Bind Plus Sepharose beads for 2 hours at 4° C. The beads were spun down and the cell lysate was then aliquoted into Eppendorf tubes containing Gamma Bind Plus Sepharose beads that had been pre-incubated with hybridoma supernatant. The tubes were rotated overnight at 4° C. After extensive washing, bound antigen was eluted from the beads by boiling in the presence of 5% wt/vol SDS, 125 mM Tris-HCl, pH 6.8, and 4% vol/vol β-mercaptoethanol, and 50% vol/vol glycerol. Proteins were then subjected to SDS-PAGE. After electrophoresis, the gel was fixed for 30 minutes with 60% H₂O/30% methanol/10% acetic acid. The gel was then washed for 30 minutes with water then dried down. The dried gel was put on film (Kodak® Biomax MS® film with appropriate Biomax MS® screen) overnight.

[0098] f. Soft Agar Colony Formation Assay

[0099] For anchorage-independent cell growth, a soft agar colony formation assay was performed in a six-well plate. Each well contained 2 mL of 1% agar in complete medium as the bottom layer. The top layer contained 2 mL 0.5% agar in complete medium, 1000-10000 LNCaP cells, and 5 μg/mL mAb (anti-ATP synthase, anti-NCAM2, or anti-Trop1). One mL complete medium was added and the cultures were maintained at 37° C. in a humidified 5% CO₂ atmosphere for up to 20 days. One mL complete medium was added once a week. Media was removed and the colonies were stained with 0.005% crystal violet in PBS for 2 hours. The number of colonies was determined by counting them under an inverted phase-contrast microscope at 100×, and a group of 10 or more cells were counted as a colony.

[0100] g. LNCaP Proliferation

[0101] LNCaP cells were plated at 20,000 cells/well into a 96 well tissue culture plate. After cells were allowed to grow undisturbed for two days, antibodies (5 μg/ml anti-ATP synthase, anti-NCAM2, anti-Trop-1, or anti-MHC class II) were added and incubated with the cells for 24 hours. Cell proliferation was measured using the AlamarBlue vital dye indicator assay. AlamarBlue reagent was added to each well and the plates were incubated for 3 to 4 hours at 37° C. to allow for fluorescence development. Fluorescence was detected at λex=530 nm, λem=590 nm. Data is expressed as the mean +/− SEM of 4 replicates.

[0102] Results and Discussions

[0103] One BALB/c mouse was immunized with a lipid raft preparation from the prostate cancer cell line LNCaP. After two boosts, lymphocytes were isolated from the mouse lymph nodes and fused with myeloma NSO cells to generate hybridomas. A total of about 700 hybridomas were generated and supernatant from each hybridoma was screened by flow cytometry for binding to LNCaP. About 203 supernatants tested positive, and they were further tested for binding to normal prostate cells. Thirty-four of these supernatants tested negative for binding to normal prostate cells. Antibodies from these 34 hybridomas were further tested against multiple cancer lines by flow cytometry to determine whether they are LNCaP specific, prostate cancer specific, or pan-cancer specific. The cancer cell lines we used include: DU 145 (prostatic), PC-3 (prostatic), PANC-1 (pancreatic), RT4 (bladder), HT-29 (colorectal), NCI-H292 (lung), T-47D (breast), and NIH:OVCAR-3 (ovarian). In addition, a primary, non-transformed HUVEC line (human umbilical vein endothelial cells) was used to ensure that these antibodies do not cross-react with normal endothelial cells. The 34 LNCaP reacting hybridomas can be divided into two main groups based on their antigen expression profiles: antibodies from 20 of these hybridomas (Table 2) showed that their antigens are expressed only in a limited number (1-3) of cancer cell lines and 14 (Table 3) showed that their antigens are expressed in multiple lines. A flow chart describing how these tumor-associated, LNCaP lipid raft-derived antigens were identified by the hybridoma technology is shown in FIG. 1.

[0104] We performed an immunoprecipitation experiment to determine the molecular weight of the antigens that showed limited expression profiles. Of the twenty antibodies used, three predominant antigens of MW 98 (recognized by 3 antibodies), 100 (recognized by 6 antibodies), and 120 kD (recognized by 4 antibodies) were identified (FIG. 2, see also the last column of Table 1). Seven antibodies were not able to immunoprecipitate antigen for molecular weight determination. The antigen grouping results indicate the hybridomas of Table 1 covered a minimum of four to a maximum of ten antigens-.

[0105] a. Lipid Raft Tumor-associated Antigen NCAM2

[0106] The identity of the antigen defined by the hybridoma P3-53 had been determined. The monoclonal antibody produced by hybridoma P3-53 (Table 1) immunoprecipitated a major protein band of 120 kD and a minor band of 110 kD (see FIG. 2, Lane 3). The P3-53 antigen is expressed only in the prostate cell line LNCaP and breast cell line T47D. The monoclonal antibody from P3-53 was conjugated to CNBr-activated Sepharose to generate an affinity column (20 mg conjugated to a 2 ml column). LNCaP whole cell lysate was prepared from 2×10⁸ cells as described in MATERIAL AND METHODS and passed onto the P3-53 affinity column. After extensive washing, the retained protein was eluted with low pH buffer. About 5 μg of the P3-53 antigen was purified. SDS-PAGE analysis followed by silver staining revealed the P3-53 antigen consisted of a major protein band of 120 kD and a minor band of 110 kD. The purified antigen was subjected to microsequencing analysis and the result showed that it had a NH2-terminal sequence of X-L-QV-T-I-S-L-S-K, where X was probably “L”, but might also be “G”. This sequence was searched against the entire NCBInr database using Protein Prospector. Only one human protein with the NH₂-terminal sequence of L-L-Q-V-T-I-S-L-S-K matched the determined P3-53 antigen sequence. The human protein is called neural cell adhesion molecule 2 (NCAM2, NCBI protein accession number 4758764, see also Paoloni-Giacobino et al, Genomics 43, 43-51 (1997)), which is a homologue of a murine protein called Rb-8 neural adhesion molecule (RNCAM, NCBI protein accession number 3334269, see also Alenius, M. and Bohm, S., J. Biol. Chem. 272, 26083-26083 (1997) and Yoshihara et al. J. NeuroSci. 17: 5830-5842 (1997). The identification of the antigen recognized by P3-53 was confirmed to be NCAM2 by MALDI-TOF peptide-mass profiling as described in the MATERIALS AND METHODS in Example 1.

[0107] The sequence of RNCAM predicted molecules having an extracellular region of 5 immunoglobulin C2-type domains followed by two fibronectin type III domains. Alternative splicing of the NCAM2 and RNCAM transcripts generate two isoforms: the long form containing a transmembrane domain and the short form containing a glycosylphosphatidylinositol-anchor attached to the membrane. The expression of RNCAM is restricted to the olfactory neurons in the brain and in the nasal vomeronasal organ. The transcript of RNCAM is not detectable in lung, gut, liver, heart, testis and kidney. The function of NCAM2 or RNCAM is not known, but the molecule may play a role in selective axon projection. NCAM2 was also shown to be a homophilic adhesion molecule (see Yoshihara et al. J. NeuroSci. 17: 5830-5842 (1997)). Thus we found that certain prostate and breast cancer cell lines express this protein marker that is neural in origin. Accordingly, because NCAM2 is expressed in a substantial percentage of prostate and breast cancer cells, an antibody to NCAM2 provides an effective treatment for prostate or breast cancer. Antibodies are better drugs than small molecules against cancer cells expressing neural markers because they do not cross the blood-brain barrier to potentially have toxic effects on normal neurons.

[0108] b. Inhibition of Prostate Cancer Cell Proliferation by Anti-NCAM2 Antibodies

[0109] As shown in Table 2 and FIG. 2, P3-53, P9-64, P10-28, and P10-29 all immunoprecipitated an antigen with a molecular weight of 120/110. The P3-53 antigen was identified as NCAM2. We tested the anti-cancer activity of 4 anti-NCAM2 antibodies in a proliferation assay using the prostate cancer cell line LNCaP. The results are shown in FIG. 3. One of the 4 antibodies, namely P9-64, had significant inhibition activity against proliferation of LNCaP cells. This reduction in cellular proliferation by P9-64 was about 39%. P10-28 and P10-29 had about 16 and 11% inhibition activity, respectively, and P3-53 was not effective. . This substantial inhibition of cell proliferation by some anti-NCAM2 antibodies suggests a potential of anti-ATP synthase antibody as an anti-tumor agent for treating prostate cancer.

[0110] c. Inhibition of Prostate Cancer Cell Colony Formation by Anti-NCAM2 Antibodies

[0111] Transformed cancer cells are resistant to anchorage-independent growth inhibition and are able to growth in soft agar without attaching to cell matrix. Formation of colonies (three-dimensional growth under tissue culture growth conditions) of cancer cells in soft agar is often correlated to the aggressiveness of the tumor in vivo. To assess whether the four anti-NCAM2 antibodies any anti-cancer activity, we used them inhibit LNCaP colony formation in vitro. As shown in FIG. 4, LNCaP cancer cell colony formation can be reduced by some anti-NCAM2 antibodies at 5 μg/ml. As in the proliferation assay, the most potent inhibitor is P9-64. It inhibited colony formation by 42%. P10-28 and P10-29 inhibited about 22% and 36% respectively. The inhibitory activity of P3-53 was again low, at about 10% inhibition. The substantial inhibition of colony formation by some anti-NCAM2 antibodies suggests a potential of anti-NCAM2 antibody as an anti-tumor agent for treating prostate cancer.

[0112] The above experiments demonstrate that NCAM2 expression is closely linked to some types of prostate cancer cells. It is localized on the surface of cancer cells, and may play a key role in the biological activities of prostate cancer cells. Blocking the activity of NCAM2 by antibodies inhibits prostate cancer cell growth, suggesting the possibility of clinical application of NCAM2 inhibitors as anti-tumor agents for treating prostate cancer.

Example 2

[0113] This example describes inhibition of tumor adhesion and spreading by cell adhesion and migration assay

[0114] Antibody inhibition of adhesion and spreading is evaluated. Tissue culture 12-well plates were coated 2 hours at room temperature with components of the extracellular matrix, i.e. vitronectin (VN), fibronectin (FN), collagen type I, type III and type IV, laminin (LA), or hyaluronic acid (HA) in Hanks buffered salt solution (HBSS). Plates are blocked for 2 hours with 1% BSA in PBS. Cells are plated in HBSS with 1 mM CaCl₂ and 1 mM MgCl₂ in the presence or absence of antibody. Cells are allowed to spread for 30 minutes to 2 hours at 37° C. prior to photography.

[0115] Inhibition of cancer cell migratory activity of anti-tumor agents is evaluated in a matrigel assay. Membranes with a pore size of 8 μm were coated with 50 μl matrigel. The membranes were inserted into 24 well plates that contain medium without supplements. Cancer cells are resuspended in medium with 10% FCS in the presence or absence of antibodies and then seeded on the matrigel coated transwell plates. Plates are incubated for 48 hours at 37° C. Thereafter, cells at the bottom of the chamber are counted using an inverted microscope.

Example 3

[0116] This example describes selection of anti-lipid raft antibodies for cancer therapy based on their antigen expression profiles and anti-cancer activities in vitro.

[0117] For solid tumors, monoclonal antibodies against the identified antigens are used to stain by immunohistochemistry normal or neoplastic human tissues to establish the expression profiles of the tumor associated antigens. Valuable tumor-associated tumor antigens should have low or no expression in normal tissues and high expression in cancer cells. To be a good targets for antibody therapy, tumor associated antigens should be differentially expressed in substantial percentage (20% and above) of certain cancer type. Valuable antibodies against these antigens may have anti-cancer activities in vitro. These activities include inhibition of cell proliferation, induction of apoptosis and inhibition of cell migration.

[0118] For hematological malignancies, monoclonal antibodies against the identified antigens are used to stain by flow cytometry patient's leukemic cell as well as normal human blood and bone marrow cells. Antigens that are expressed in hematopoietic stem cells (within the CD34-positive population), T cells, platelets, or granulocytes should be excluded because triggering or killing of these cells by antibodies will cause severe toxicity in humans. Antigens of interest may be expressed in B cells, macrophages or monocytes but not in other normal tissues. The ideal tumor-associated antigens are the ones that can be triggered to induce cell death in leukemic cells.

Example 4

[0119] This example describes the treatment of prostate cancer by antibodies specific for NCAM2 in well-established androgen-dependent and androgen-independent prostate cancer xenografts.

[0120] Six to ten week old male nude NCR nu/nu mice are inoculated subcutaneously in the mid-scapular region with 5×10⁶ androgen-dependent LNCaP cells. Cells that are injected are reconstituted with basement membrane in the form of Matrigel as described (Sato et al Cancer Res. 5, 1584-1589 (1997)). To maintain serum testosterone levels, male mice are implanted with 12.5 mg sustained release testosterone pellets subcutaneously prior to receiving the tumor cell inoculation. Antibodies specific for NCAM2 are given intraperitoneally on day 2 and 4. Tumors are measured every three to four days with vernier calipers. Tumor volumes are calculated by the formula π/6×(larger diameter)×(smaller diameter)². For the androgen-independent prostate cancer xenograft studies, DU 145 or PC-3 cells are used.

Example 5

[0121] This example describes the human prostate cancer therapeutic regime by using the inhibitors NCAM2.

[0122] The patient's cancer biopsy sample is stained positively for the expression of NCAM2 on the cancer cell surface by immunohistochemistry for the patient to be eligible for treatment. Anti-NCAM2 antibodies are administered either intravenously or subcutaneously in a dose range from 0.05 to about 25 mg/kg. Patients receive at least 4 weekly doses. Tumor size is monitored by CAT scan or MRI prior to therapy and post therapy. Reduction of the tumor size is the primary indication of the drug's efficacy. Tumor shrinkage by 50% or more is considered as a partial response. Complete disappearance of the tumor is considered as a complete response. For prostate cancer patients, PSA level prior to therapy and post therapy is also monitored as a secondary indication of treatment efficacy.

[0123] Although the invention has been described with reference to the presently preferred embodiments, it should be understood that various modifications can be made without departing from the spirit of the invention.

[0124] All publications, patents, patent applications, and web sites are herein incorporated by reference in their entirety to the same extent as if each individual patent, patent application, or web site was specifically and individually indicated to be incorporated by reference in its entirety. 

1. A method of treating prostate cancer in a subject in need of the treating comprising administering to said subject an inhibitor of NCAM2 or RNCAM in a pharmaceutically effective amount.
 2. The method according to claim 1, wherein said inhibitor is a protein that directly interacts with NCAM2.
 3. The method according to claim 1, wherein said inhibitor down-regulates biological activities of NCAM2.
 4. The method according to claim 1, wherein said inhibitor inhibits protein expression of NCAM2.
 5. The method according to claim 4, wherein said inhibitor is an anti-sense nucleic acid of a nucleic acid sequence encoding part or full NCAM2.
 6. The method according to claim 4, wherein said inhibitor is a transcriptional factor.
 7. The method according to claim 2, wherein said inhibitor is an antibody that binds to NCAM2 or neutralizes biological activities of NCAM2.
 8. The method according to claim 7, wherein said antibody inhibits prostate cancer cell proliferation by at least 10%.
 9. The method according to claim 7, wherein said antibody inhibits prostate cancer cell proliferation by at least 35%.
 10. The method according to claim 7, wherein said antibody is a monoclonal antibody.
 11. The method according to claim 10, wherein said monoclonal antibody is a humanized antibody or a fully human antibody.
 12. The method according to claim 10, wherein said monoclonal antibody is a chimeric antibody.
 13. The method according to claim 10, wherein said antibody is an antibody tetramer, Fab, (Fab′)₂, or Fv.
 14. The method according to claim 1, wherein said inhibitor is an antibody conjugate comprising an anti-NCAM2 antibody.
 15. The method according to claim 14, wherein said anti-NCAM2 antibody is conjugated to a cytotoxin agent.
 16. The antibody conjugate according to claim 15, wherein said cytotoxin agent is a protein cytotoxin or a Fc domain of a monoclonal antibody.
 17. The method according to claim 1, further comprising administering a chemotherapeutic agent to the subject, wherein said treating is formulated in a manner allowing it to be administered serially or in combination with another agent for treatment of cancer.
 18. The method according to claim 1, further comprising administering radiation therapy to the subject, wherein said treating is formulated in a manner allowing it to be administered serially or in combination with another agent for treatment of cancer.
 19. A method of detecting prostate cancer comprising detecting presence of NCAM2 in prostate cells of a subject in need of the detecting.
 20. A method of detecting breast cancer comprising detecting presence of NCAM2 in breast cells of a subject in need of the detecting.
 21. The method according to claim 19 or claim 20, wherein said detecting comprising using an antibody conjugate, wherein said antibody conjugate comprises an antibody or an antibody fragment that binds to at least one epitope of human NCAM2, wherein said antibody or antibody fragment is conjugated to a diagnostic imaging agent.
 22. A pharmaceutical composition comprising an inhibitor of NCAM2 and a pharmaceutical carrier.
 23. The pharmaceutical composition according to claim 22, wherein said inhibitor is an anti-NCAM2 antibody.
 24. The pharmaceutical composition according to claim 23, wherein said anti-NCAM2 antibody is a humanized or human anti-NCAM2 antibody.
 25. An antibody that binds to or neutralizes NCAM2.
 26. The antibody according to claim 25, wherein said antibody inhibit tumor cell proliferation by more than 10%
 27. The antibody according to claim 25, wherein said antibody inhibit tumor cell proliferation by more than 30%
 28. The antibody according to claim 25, wherein said tumor cell is a prostate cancer cell.
 29. The antibody according to claim 25, wherein said antibody inhibits prostate cancer cell colony formation.
 30. The antibody according to claim 25, wherein said antibody inhibits prostate cancer cell colony formation by more than 20%.
 31. The antibody according to claim 25, wherein said antibody inhibits prostate cancer cell colony formation by more than 40%.
 32. The antibody according to claim 25, wherein said antibody is a humanized anti-NCAM2 antibody.
 33. The antibody according to claim 25, wherein said antibody is a fully human anti-NCAM2 antibody.
 34. The antibody according to claim 25, wherein said antibody is an antibody tetramer Fab, (Fab′)₂, or Fv.
 35. A method of treating breast cancer in a subject in need of the treating comprising administering to said subject an inhibitor of NCAM2 or RNCAM in a pharmaceutically effective amount.
 36. A hybridoma cell line P9-64 deposited with American Type Culture Collection (ATCC) as accession number PTA-4734.
 37. A monoclonal antibody produced by the hybridoma cell line according to claim
 36. 38. A method of inhibiting growth of a cancer cell comprising contacting an inhibitor of NCAM2 with said cancer cell.
 39. The method according to claim 38, wherein said cancer cell is a prostate cancer cell.
 40. The method according to claim 38, wherein said inhibitor is an antibody that binds to or neutralizes NCAM2.
 41. The method according to claim 40, wherein said antibody inhibits prostate cancer cell proliferation.
 42. The method according to claim 41, wherein said antibody inhibits prostate cancer cell proliferation by at least 30%. 